Ultrapurification of factor VIII using monoclonal antibodies



10-20 units of VIII:C per ml of eluant are obtained with the presentinvention, in contract to 0.5-1.25 units per ml of eluant with theTuddenham et al method.

The present method also permits the selection of a monoclonal antibodyhaving a high affinity for VIII:RP; however, the use of polyclonalantibodies results in varying affinities. It should be realized thatthere is an indirect relationship between the affinity of the boundantibody for VIII:RP and the elution of VIII:RP. Thus, the higher theaffinity of the antibody for VIII:RP, the less VIII:RP will be presentwith VIII:C in the eluant. The present invention also makes it possibleto produce an unlimited supply of the specified monoclonal antibody,thus eliminating variations among different batches.

Although Austen, as earlier described, has reported the use ofaminohexyl-agarose to separate VIII:C from VIII:RP, such a material hasnot heretofore been used to concentrate VIII:C following a separationand purification step. Heretofore, the highest VIII:C concentrationsachieved by using aminohexyl agarose in chromatography were 0.53 unitsper ml of eluant for human protein and 2.38 per ml of eluant for porcineVIII:C. The present method permits concentrations several orders ofmagnitude greater than these. Perhaps of even greater significance, isthe fact that the present invention provides for a greater purificationof human VIII:C than has ever been reported (164,000 vs 17,000 fold overplasma). The present method, which is described in more detailhereinafter, yields VIII:C with a specific activity of 2,300 units/mgwhen commercial concentrate is used. This corresponds to a 164,000 foldpurification from plasma. The ratio of VIII:C to VIII:RP is greater than10⁵ as compared to the ratio in plasma.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following description provides details of the manner in which theembodiments of the present invention may be made and used in order toachieve the separation, purification and concentration of VIII:C to adegree of purity and concentration not known heretofore. Thisdescription, while exemplary of the present invention, is not to beconstrued as specifically limiting the invention and such variationswhich would be within the purview of one skilled in this art are to beconsidered to fall within the scope of this invention.

A. Preparation of Monoclonal Antibody to VIII:RP

The monoclonal antibody to VIII:RP which is subsequently bound to theseparation substrate may be prepared in a stepwise procedure startingwith a highly purified preparation of factor VIII/von Willebrand factor(VIII:C/VIII:RP complex). The purification for immunization isaccomplished with material obtained from a plasma source. Less highlypurified material for coating polyvinyl plates is obtained in higherconcentration from commercial extracts such as FACTORATE (trademark ofArmour Pharmaceutical Co., Tuckahoe, N.Y.) or Hemophil (trademark ofHyland Laboratories, Costa Mesa, California). Purification is performedby a standard agarose-gel filtration of cryoprecipitate, such as thatdescribed by Zimmerman and Roberts, "Factor VIII Related Antigen",appearing in IMMUNOASSAYS: CLINICAL LABORATORY TECHNIQUES FOR THE1980's, R. M. Nakamura et al, eds., Alan R. Liss, Inc., New York, pp.339-349 (1980). Mice were injected with highly purified factor VIII/vonWillebrand factor obtained from plasma according to the followingprocedure. On day zero, the mice are injected intraperitoneally with acomposition prepared by dissolving (or suspending) 10 Mg of the proteinin 0.1 ml of buffer containing 0.05 M Tris, 0.15 M sodium chloride, 0.02% sodium azide, 1 mM phenyl methyl sulfonyl fluoride, traysylol 10units/ml at pH7.3. and shaking with an equal volume of complete Freund'sadjuvant. On day 14, the mice are again injected with the same materialexcept that incomplete Freund's adjuvant is substituted for completeFreund's adjuvant. On day 21, the injection of day 14 is repeated. Onday 38, the mice are injected with purified VIII:C/VIII:RP only. On day42, the spleens of the mice are removed and fused according to astandard procedure, of the type described by J. P. Brown et al "ProteinAntigens of Normal and Malignant Human Cells Identified byImmunoprecipitation with Monoclonal Antibodies", JOURNAL OF BIOLOGICALCHEMISTRY, Vol. 225, pp. 4980-4983 (1980). The standard technique isvaried only to the extent that 35% polyethylene glycol 1000 issubstituted for 50% polyethylene glycol. A radioimmunoassay method forclones producing antibody to VIII:RP is performed according to thefollowing procedure. Polyvinyl plates with a "V" bottom, flexible typeare coated with 0.1 ml of factor VIII purified from commercial extractaccording to the procedure indicated above and having a concentration of0.125 mg/ml of protein. The plates are blocked with albumin, washed withbuffer and incubated with the culture fluids from the clones to betested. The plates are then washed and reacted with rabbit anti-mouseIgG antiserum, washed a second time and ¹²⁵ I labeled goat anti-rabbitIgG antiserum is added to the wells and incubated. The plates are againwashed, then dried and the wells cut-out and counted. After determiningthe clones which are positive they are subcloned at least twice andstable clones producing antibody to VIII:RP are then injected into theperitoneal cavities of Balb/C mice which have been pretreatedintraperitoneally with 0.5 ml of pristane at least four days prior toinjection of cells. Hybridoma cells are injected at concentrations ofapproximately 5×10⁶ cells per mouse in 0.5 ml of Delbecco's modifiedEagle's medium without fetal bovine serum. The mice are tapped whenbloated and ascites fluid is collected in heparin at approximately 10units/ml. Ascites fluid from multiple mice is pooled to provide aconvenient volume for subsequent isolation of the monoclonal IgG. If theheparinized ascites fluid is not used immediately, it may be stored at-70° C. and thawed just prior to use. The final yield of IgG from theascites fluid is approximately 1 g of IgG per 100 ml of ascites fluid.

The specificity of the monoclonal IgG for the purpose of purifyingVIII:C may be assessed by coupling the IgG to a separation substratemedium, in the manner described hereinafter, and demonstrating that thebound IgG removes from VIII:RP and VIII:C from plasma and that theVIII:C may be subsequently eluted with a solution containing calciumions while the VIII:RP remains complexed to the monoclonal IgG which isbound to the solid-state substrate.

The monoclonal IgG, which is to be used subsequently to prepare theimmunoadsorbent, may be isolated from heparinized pooled ascites fluidimmediately after collection or a frozen portion of the stored solutionmay be thawed. Regardless of whether fresh or frozen material is used,the solution is brought to 4° C. and treated with an equal volume ofphosphate buffered saline solution (PBS), the composition of which isset forth below. The diluted ascites is precipitated by dropwiseaddition with stirring at 4° C. of an equal volume of saturated ammoniumsulfate (SAS); prepared by boiling an excess of ammonium sulfate inwater, cooling to 4° C., filtering undissolved crystals and adjustingthe pH to 7.0 with ammonium hydroxide. The precipitate and itssupernatant liquid are stirred for at least 2 hours and centrifuged at4° C. Centrifugations are preferably carried out at 14,000 rpm for 60minutes (30,000×g). The supernatant solution of ascites is precipitatedtwice more with SAS and the mixture of precipitate and supernatantliquid stirred and centrifuged in the same manner as in the first cycle.The pellets resulting from the third precipitation are resuspended in avolume of PBS equal to that of the diluted ascites fluid and thendisalyzed exhaustively against PBS. Clots appearing in the dialysis bagsare removed by centrifugation at 20° C. The dialyzed IgG is adsorbed bystirring it with a 5% aqueous solution of aluminum hydroxide at roomtemperature and centrifuging at 20° C. after adsorption. The adsorptiontreatment is repeated at least three more times using 2.5% aluminumhydroxide solution for each treatment after the first. The adsorbed IgGis brought to 4° C. and reprecipitated once with SAS as described above.The precipitated pellets may be stored at -20° C. until used.

B. Preparation of the Immunoadsorbent

The immunoadsorbent is prepared by suitably preparing the monoclonal IgGfor coupling, preparing the solid substrate for coupling and reactingthe two components to bind the former to the later.

(i) Preparation of IgG for Coupling

Either freshly precipitated IgG may be used or previously frozenprecipitate may be thawed for use. The material is then dialyzed againstPBS, and while still in the PBS, the volume and IgG concentration (A₂₈₀/1.4=mg/ml IgG) are determined. The IgG is then treated with between 10and 30 microliters, preferably 20 microliters, ordiisopropylfluorophosphate per 50 ml of IgG solution. The resultingsolution is stirred at room temperature in a hood for 30 minutes and thetreated IgG, immediately prior to use, is dialyzed overnight againstcoupling buffer. The coupling buffer found most suitable is a 0.25 Msodium bicarbonate solution adjusted to a pH of 9, preferably withsodium hydroxide.

(ii) Preparation of Solid Substrate for Coupling

Although the monoclonal antibody may be bound to any material which doesnot have a high affinity for protein, particularly factor VIII itself,such materials as glass beads, agarose and derivatives thereof arepreferred. Most preferred is a crosslinked agarose availablecommercially as a gel known as Sepharose CL2B (trademark of PharmaciaFine Chemicals, Piscataway, N.J.).

The method of preparing the preferred immunoadsorbent resin is generallythe same as that disclosed in the literature, such as the method of J.Porath et al, JOURNAL OF CHROMATOGRAPHY, Vol. 86, pp. 53-56 (1973). Themethod found most suitable is as follows: a volume of about 2 liters ofSepharose CL2B is placed in an acid-cleaned 2 liter sintered glassfilter funnel. The resin is washed with water and filtered to a moistcake. The washed resin is placed in a large (approximately 4 liter)glass beaker equipped with a magnetic stirring bar. To the resin is thenadded 750 ml of cold potassium phosphate buffer solution, prepared bymixing one part of a 5 M dibasic potassium phosphate solution with twoparts of 5 M tribasic potassium phosphate solution. Sufficient coldwater is added to bring the final volume to 3 liters. The mixture isthen chilled to 4° C. and maintained at between 4°-10° C. in anice-water bath placed on a magnetic stirring plate. In a hood, cyanogenbromide is added to 300 ml of water in a stoppered glass bottlecontaining a magnetic stirring bar. The mixture is rapidly stirred untilsolution results. The cyanogen bromide solution is then added withstirring over a 2 minute period to the cold Sepharose mixture. Stirringis continued for an additional 8 minutes and then transferred to achilled 2 liter sintered glass filter funnel supported in a 4 litervacuum flask. The cyanogen bromide treated resin is then washed withapproximately 20 liters of cold water or until the pH of the filtrate isneutral. The washed resin is then quickly equilibrated with coldcoupling buffer and then transferred to a 4 liter plastic beakerequipped with a large magnetic stirring bar.

(iii) Coupling the Monoclonal Antibody to the Solid Substrate

The solid substrate resin, prepared as indicated above, is ready to beused when it is equilbrated with coupling buffer and should not bestored thereafter. Accordingly, the resin mixture is combined with theIgG which was previously dialyzed overnight against coupling buffer. Thecombined resin/IgG suspended mixture is stirred at 4° C. for a period ofabout 24 hours. The A₂₈₀ of an undiluted sample of the supernatantcoupling liquid may be determined using bovine serum albumin (BSA) as astandard or Bio-Rad protein assay (Bradford reagent) with BSA asstandard. The percentage ligand which is coupled may then be calculated.When the above described procedure is followed, this is usually about95%. Any remaining active sites on the resin not coupled to antibody maybe blocked by washing the resin on a sintered glass filter funnel withcold coupling buffer containing 0.1 M glycine. The resin is thenresuspended in this solution to a final volume equal to that when theresin and antibody, each in coupling buffer, were combined. Thesuspension is stirred slowly overnight at 4° C. The resin is then washedthoroughly with VIII:C-buffer, the composition of which is given below.The coupled, blocked resin is then pre-eluted with VIII:C-bufferadditionally containing 0.5 M calcium ions, preferably calcium chloride.The resin is again washed with VIII:C buffer alone and stored at 4° C.or in a continuously pumped column at room temperature until ready foruse. The coupling density of IgG to SEPHAROSE should be 2-5 g,preferably 3-4 g IgG/liter of SEPHAROSE.

C. Separation and Purification of VIII:C

(i) Sample preparation of factor VIII, such as human and animal plasmasand commercial concentrates of factor VIII, may be employed in thepresent invention and the method is not limited as to a particular typeof material. Preferred materials, and those which have demonstratedsuccessful results, are porcine and human plasmas and commerciallyavailable concentrates of human factor VIII, such as FACTORATE availablefrom Armour Pharmaceutical Co. The following description providesdetails for using both porcine plasma or commercial human concentratesuch as FACTORATE:

FACTORATE is reconstituted by adding 25 ml portions of VIII:C-buffer tothe contents of each of 20 bottles containing 400-500 VIIIC units perbottle (25 ml per bottle). The mixture is adjusted to a final volume of1 liter with VIII:C-buffer. A sample aliquot of 0.5 ml may be removedfor assay and the remaining material applied to the immunoadsorbentcolumn overnight at a rate of approximately 60 ml/hour.

Porcine plasma, when not freshly drawn, is citrated by conventionalmeans and stored frozen. When ready to be used it is thawed at atemperature of between 35°-40° C., preferably 37° C. and applieddirectly to the column at 60 ml/hour.

It should be noted that while the description of the present inventionrefers, and is directed primarily, to the use of immunoadsorbent coupledparticles in a chromatography column, it is within the scope of thisinvention to perform batchwise separations by placing the antibody-boundresin particles in a suitable container and after adding reconstitutedconcentrate or plasma, VIII:C as outlined above and described in moredetail below.

When the process is carried out in a chromatography process, thefollowing embodiments are preferred:

The resin is placed in a column, such as an Amicon 86001, (trademark ofAmicon Corp., Lexington, Mass.), equipped with a peristaltic pump and ahigh flow head. When concentrate is used as the source of factor VIII,for 20 bottles of diluted concentrate, approximately 1.5 liters ofresin, prepared as indicated above, is used. When porcine plasma isused, 150 ml of resin is used for each liter of plasma.

After the sample is applied to the column, it is washed with 1 liter ofVIII:C-buffer, followed by a second washing with VIII:C-buffer whichadditionally contains 0.5 M NaCl. Approximately 20 liters ofsaline-buffer is used when factor VIII is applied as concentrate and 20bed volumes when porcine plasma is employed. Optimum results areobtained with a flow rate of 1 liter/hour.

Elution of purified VIII:C is accomplished with VIII:C-buffer containingcalcium ions. Although a linear gradient, as taught by Tuddenham et al,supra, works well, it is not required in order to accomplish the objectof this invention; a solution having a fixed calcium ion concentrationis quite adequate. Thus, when VIII:C derived from concentrate is beingeluted, VIII:C-buffer, 0.25 to 0.5 M with respect to calcium chloride,preferably 0.35 M, is used advantageously as a flow rate of between 450to 750 ml/hour and preferably 600 ml/hour. When the VIII:C is obtainedfrom porcine plasma, elution is performed with VIII:C-buffer being acalcium chloride concentration of between 0.35 and 0.7 M, preferably 0.5M and at a flow rate of between 10 and 30 ml/hour, preferably 20ml/hour. Fractions of 12 ml and 3 ml are collected for VIII:Coriginating from concentrate and porcine plasma, respectively. Thosefractions containing at least 1.0 unit/ml of VIII:C activity are pooledand the total volume and activity of the pool determined.

The VIII:C pool is initially concentrated to 10-20 ml by a standardprocedure such as pressure ultrafiltration. For this purpose, Amiconstirred cell in which a YM-10 membrane under 50 psi of nitrogen pressurehas been found to work well. Slow stirring is continued for 30 minutesafter nitrogen pressure is released, and the volume and activity of theconcentrated pool are determined. The pool may be stored for a briefperiod, that is, overnight for example, if a temperature of 4° C. ismaintained.

It may be noted that the immunoadsorbent column described above may beregenerated by treatment of the column with 2 bed volumes of 3 M aqueoussodium thiocyanate solution run at a flow rate of about 0.5-1 liter/hourto elute VIII:RP.

D. Concentration of Purified VIII:C

Although the VIII:C recovered from the separation from VIII:RP by meansof the immunoadsorbent column is highly purified, it is still too diluteto be therapeutically useful. Further concentration and a four foldincrease in purification when porcine plasma is used is accomplished byuse of an aminohexyl agarose column which is prepared and used in thefollowing manner:

(i) Preparation and/or Conditioning of a Aminohexyl Agarose Column:

Aminohexyl agarose is agarose which has been reacted with1,6-diaminohexane to yield an agarose resin having a number of 6 carbonatom chains, each of which has a terminal amino group. It may beprepared according to the method described by Austen, supra, or acquiredfrom a commercial supplier. One such material which has been usedsuccessfully in the present invention is available under the name ofAH-SEPHAROSE 4B (trademark of Pharmacia Fine Chemicals, Piscataway,N.J.).

Whether prepared or purchased, the resin should be conditioned prior touse. This may be accomplished as follows, the volumes, amounts anddimensions being adjusted in proportion to the amount of material to beconcentrated:

Approximately 1 gram of aminohexyl agarose (AH-SEPHAROSE 4B) is placedin a sintered glass filter funnel and washed with at least 200 ml of 0.5M sodium chloride, while stirring. The resin is then equilibrated withVIII:C-buffer and packed in a column of approximately 0.9 cm diameter. ABio-Rad Econo-Column with flow adapters has proven quite suitable forthe type of use considered here. The bed volume of the packed column isapproximately 4 ml.

(ii) Application to and Use of the Aminohexyl Agarose Column

The concentrated pool, prepared as described above, is diluted 1:10 inVIII:C-buffer to a final concentration of 100-200 ml when using theamounts of resin and column size as described in the immediatelypreceding section. The diluted pool is applied to the column at a flowrate of 200 ml/hour.

The column is then washed with VIII:C-buffer which contains calciumions, preferably from calcium chloride. The solution should be between0.01 M to 0.03 M, preferably 0.025 M with respect to calcium ions.

Elution of the concentrated VIII:C is achieved at a flow rate of between5 to 20 ml/hour, preferably 10 ml/hour with VIII:C-buffer containing ahigher concentration of calcium ions than was employed with thepreceding washing step. Again, calcium chloride is the preferred sourceof calcium ions in a concentration of between 0.25 to 0.5 M, preferably0.3 M. Fractions of 1 ml volume are collected and assayed as describedbelow. Collected fractions may be stored at 4° C. or frozen.Preparations of VIII:C obtained from a porcine plasma source should bestabilized within 5 to 10% human serum albumin prior to storage.

Assays may be performed by diluting the fractions with VIII-C buffer ifnecessary and further diluting the fraction 1:100 in assay buffer priorto addition to the substrate. A standard partial thromboplastin timeassay is employed.

The composition of the buffer solutions is as follows:

Phosphate Buffered Saline Solution:

1.6 g sodium phosphate, monobasic monohydrate

8.4 g sodium phosphate, dibasic anhydrous

61.4 sodium chloride

Water to 7 liters

pH of buffer is 7.2

VIII:C-Buffer

ml 0.02 M imidazole

ml 0.15 M sodium chloride

ml 0.10 M lysine

ml 0.02% sodium azide

pH of buffer is adjusted with concentrated hydrochloric acid to 6.8.

The data listed hereinafter in Tables I and II are representative ofthat obtained according to the present invention, as described above.

                                      TABLE I                                     __________________________________________________________________________    VIII:C Obtained From FACTORATE Concentrate as the Source of                   VIII:C/VIII:RP                                                                                                         Specific                                                                           From                                            VIIIC                                                                             VIIIC                Activity                                                                           Plasma                                     Volume                                                                             (Units/                                                                           (Total                                                                            Protein                                                                            Protein                                                                             Recovery                                                                            (Units/                                                                            (Fold                                      (ml) ml)*                                                                              Units)                                                                            (mg/ml)                                                                            (Total mg)                                                                          (%)   mg)  Purif.)                         __________________________________________________________________________    Sample Applied to                                                                        500  18.8                                                                              9400                                                                              29   14.500                                                                              --    0.7  50                              Immunoadsorbent                                                               Pool resulting from                                                                      1020  4.6                                                                              4692                                                                              --   --    50    --   --                              Immunoadsorbent                                                               Pool After Initial                                                                        20  134 2680                                                                              --   --    29(57)                                                                              --   --                              Concentration                                                                 Sum Resulting from                                                                       --   --  1576                                                                              --   --    17(59)                                                                              --   --                              Aminohexyl Column                                                             Aminohexyl 0.95 1172                                                                              1112                                                                              0.51 0.48  12    2294 163.857                         Fraction #3                                                                   Aminohexyl --   545 --  0.23 --    --    2370 169.285                         Fraction #4                                                                   __________________________________________________________________________     *A frozen human plasma pool used as the standard for VIIIC assays and         assigned the value of 1 human unit per ml.                               

                                      TABLE II                                    __________________________________________________________________________    VIII:C Obtained From Citrated Porcine Plasma                                                                           Specific                                                                           From                                            VIIIC                                                                             VIIIC                Activity                                                                           Plasma                                     Volume                                                                             (Units/                                                                           (Total                                                                            Protein                                                                            Protein                                                                             Recovery                                                                            (Units/                                                                            (Fold                                      (ml) ml) Units)                                                                            (mg/ml)                                                                            (Total mg)                                                                          (%)   mg)  Purif.)                         __________________________________________________________________________    Sample Applied to                                                                        1000  1* 1000                                                                              76   76.000                                                                              100   0.013                                                                              --                              Immunoadsorbent                                                               Pool Resulting from                                                                       70  8.8 613 --   --    61    --   --                              Immunoadsorbent                                                               Pool After Initial                                                                       5.76 88  494.5                                                                             0.242                                                                              1.355 49.5   364  28.000                         Concentration                                                                 Sum Resulting from                                                                       5.0  49  247 0.035                                                                              0.175 25    1413 109.000                         Aminohexyl Column                                                             __________________________________________________________________________     *Porcine plasma used as the standard for VIIIC assays and assigned the        value of 1 porcine VIIIC unit per ml.                                    

Although only preferred embodiments are specifically illustrated anddescribed herein, it will be appreciated that many modifications andvariations of the present invention are possible in light of the aboveteachings and within the purview of the appended claims withoutdeparting from the spirit and intended scope of the invention.

What is claimed is:
 1. An improved method of preparing Factor VIIIprocoagulant activity protein comprising the steps of(a) adsorbing aVIII:C/VIII:RP complex from a plasma or commercial concentrate sourceonto particles bound to a monoclonal antibody specific to VIII:RP, (b)eluting the VIII:C, (c) adsorbing the VIII:C obtained in step (b) inanother adsorption to concentrate and further purify same, (d) elutingthe adsorbed VIII:C, and (e) recovering highly purified and concentratedVIII:C.
 2. A method according to claim 1, wherein the elutant used ineach of steps (b) and (d) is a saline solution.
 3. The method accordingto claim 2, wherein the saline solution is calcium chloride.
 4. Themethod according to claim 3, wherein the concentration of said calciumchloride solution used in steps (b) and (d) ranges from about 0.25 M toabout 0.5 M.
 5. The method according to claim 1, wherein said adsorbentparticles in step (a) are agarose.
 6. The method according to claim 1,wherein aminohexyl agarose is employed in step (c) as the adsorbent. 7.The method according to claim 6, wherein calcium chloride solution isemployed as the elutant in steps (b) and (d), concentration of saidcalcium chloride solution ranging from about 0.25 M to about 0.5 M instep (b) and from about 0.25 M to about 0.5 M in step (d).
 8. Animproved immunoadsorbent for isolation and purification of VIII:C fromVIII:C/VIII:RP comprising a monoclonal antibody specific to VIII:RPbound to solid particles.
 9. The improved immunoadsorbent of claim 8,wherein said solid particles comprise a resin.
 10. The improvedimmunoadsorbent of claim 9, wherein said resin comprises agarose. 11.The improved immunoadsorbent of claim 10, wherein said agarose iscross-linked agarose.
 12. The improved immunoadsorbent of claim 11,wherein said immunoadsorbent has a coupling density of 3 to 4 g ofmonoclonal antibody per liter of agarose.
 13. Highly purified andconcentrated .Iadd.human or porcine .Iaddend.VIII:C prepared inaccordance with the method of claim
 1. 14. Highly purified andconcentrated .Iadd.human or porcine .Iaddend.VIII:C prepared inaccordance with the method of claim
 6. 15. In a method for purifyingFactor VIII procoagulant activity protein from plasma or concentrate,the improvement comprising the step of passing said plasma orconcentrate through a chromatographic type column having adsorbent towhich is bound monoclonal antibodies which is specific to VIII:RP andeluting the VIII-C therefrom.
 16. The method according to claim 15,wherein said adsorbent is agarose and said elutant is a saline solution..Iadd.
 17. A VIII:C of claim 13, wherein said VIII:C is human VIII:C..Iaddend. .Iadd.18. A VIII:C of claim 14, wherein said VIII:C is humanVIII:C. .Iaddend. .Iadd.19. An improved immunoadsorbent of claim 8wherein said monoclonal antibody remains bound to VIII:RP during elutionof VIII:C. .Iaddend. .Iadd.20. An improved immunoadsorbent of claim 19,wherein said elution is carried out with a saline solution. .Iaddend..Iadd.21. An improved immunoadsorbent of claim 20 wherein said solutioncontains calcium ions. .Iaddend. .Iadd.22. An improved immunoadsorbentof claim 21, wherein said solution is calcium chloride. .Iaddend..Iadd.23. An improved immunoadsorbent of claim 22, wherein theconcentration of said calcium chloride is from 0.25 M to about 0.5 M..Iaddend. .Iadd.24. A human VIII:C preparation having a potency in therange of 134 to 1172 units per ml, and being substantially free ofVIII:RP. .Iaddend. .Iadd.25. A human VIII:C preparation of claim 24,wherein the VIII:C concentration is at least 160,000 fold purifiedrelative to VIII:C in plasma. .Iaddend. .Iadd.26. A human VIII:Cpreparation of claim 24, wherein the ratio of VIII:C to VIII:RP isgreater than 100,000 times the ratio in plasma. .Iaddend. .Iadd.27. Ahuman VIII:C preparation of claim 24, wherein said VIII:C is isolatedfrom VIII:C/VIII:RP and 90-100 percent of the VIII:RP has been removed..Iaddend. .Iadd.28. A human VIII:C preparation having a specificactivity greater than 2240 units/mg. .Iaddend. .Iadd.29. A human VIII:Cpreparation of claim 28 wherein the potency is in the range of 134 to1172 units/ml. .Iaddend. .Iadd.30. An improved method of preparingFactor VIII procoagulant activity protein comprising the steps of(a)adsorbing a VIII:C/VIII:RP complex from a plasma or commercialconcentrate source onto a substrate to which is bound a monoclonalantibody specific to VIII:RP, (b) eluting the VIII:C, (c) adsorbing theVIII:C obtained in step (b) in another adsorption to concentrate andfurther purify same, (d) eluting the adsorbed VIII:C, and (e) recoveringhighly purified and concentrated VIII:C. .Iaddend. .Iadd. A methodaccording to claim 30, wherein said substrate is a resin. .Iaddend..Iadd.32. A method according to claim 30, wherein said substrate iscomprised of particles. .Iaddend. .Iadd.33. An improved immunoadsorbentfor isolation and purification of VIII:C from VIII:C/VIII:RP comprisinga monoclonal antibody specific to VIII:RP bound to a substrate..Iaddend. .Iadd.34. Highly purified and concentrated human or porcineVIII:C prepared in accordance with the method of claim
 30. .Iaddend..Iadd.35. In a method for purifying Factor VIII procoagulant activityprotein from plasma or concentrate, the improvement comprising the stepof passing said plasma or concentrate over a substrate to which is boundmonoclonal antibodies which are specific to VIII:RP and eluting theVIII:C therefrom. .Iaddend. .Iadd.36. The method according to claim 35,wherein said substrate is agarose and said elutant is a saline solution..Iaddend.